Project Summary Tyrosine phosphorylation can regulate protein function, and this is a cornerstone of cell signaling networks. Ty- rosine phosphorylation often becomes dysregulated in cancer and therefore, understanding the effect of phos- phorylation on protein function will be paramount to identifying therapeutic interventions in cancer. Unfortunately, an important tool in the basic research of phosphorylation ? testing the effect of protein phosphorylation by com- paring the function of the phosphorylated form with an unphosphorylated form of the protein in in vitro assays ? is signi?cantly hindered by our limited knowledge in how to make a phosphorylated protein. This project seeks to build a molecular technology, inspired by previous observations of biochemistry in cell networks, to overcome this requirement and improve the pace of basic research. The goal of this research is to build a fast, accessible and inexpensive method for producing phosphorylated and soluble proteins in a bacterial system. The goals will be achieved by: development of a molecular toolkit for the enhancement and control of precise phosphorylation on protein substrates using secondary kinase-substrate targeting approaches, testing the toolkit on a set of sub- strates, and comparing the outcome with current technologies. It is anticipated that this technology will be less expensive, more physiologically-relevant, and capable of producing a larger variety of phosphorylated forms of proteins than current molecular approaches to studying the effect of protein phosphorylation.